Suppressor Mutants: History and Today’s Applications

For decades, biologist have exploited the near boundless advantages that molecular and genetic tools and analysis provide for our ability to understand biological systems. One of these genetic tools, suppressor analysis, has proven invaluable in furthering our understanding of biological processes and pathways and in discovering unknown interactions between genes and gene products.

The division defect of a Bacillus subtilis minD noc double mutant can be suppressed by Spx-dependent and Spx-independent mechanisms

During growth, bacteria increase in size and divide. Division is initiated by the formation of the Z-ring, an intense ring-like cytoskeletal structure formed by treadmilling protofilaments of the tubulin homolog FtsZ. FtsZ localization is thought to be controlled by the Min and Noc systems, and here, we explore why cell division fails at high temperature when the Min and Noc systems are simultaneously mutated. Microfluidic analysis of a minD noc double mutant indicated that FtsZ formed proto-Z-rings at periodic inter-chromosome locations but that the rings failed to mature and become functional. Extragenic suppressor analysis indicated that a variety of mutations restored high temperature growth to the minD noc double mutant, and while many were likely pleiotropic, others implicated the proteolysis of the transcription factor Spx. Further analysis indicated that a Spx-dependent pathway activated the expression of ZapA, a protein that primarily compensates for the absence of Noc. Additionally, an Spx-independent pathway increased the activity of the divisome to reduce the length of the cytokinetic period. Finally, we provide evidence of an as-yet-unidentified protein that is activated by Spx and governs the frequency of polar division and minicell formation.

Cancer LncRNA Census 2 (CLC2): an enhanced resource reveals clinical features of cancer lncRNAs

Long non-coding RNAs (lncRNAs) play key roles in cancer and are at the vanguard of precision therapeutic development. These efforts depend on large and high-confidence collections of cancer lncRNAs. Here, we present the Cancer LncRNA Census 2 (CLC2). With 492 cancer lncRNAs, CLC2 is 4-fold greater in size than its predecessor, without compromising on strict criteria of confident functional/genetic roles and inclusion in the GENCODE annotation scheme. This increase was enabled by leveraging high-throughput transposon insertional mutagenesis screening data, yielding 92 novel cancer lncRNAs. CLC2 makes a valuable addition to existing collections: it is amongst the largest, contains numerous unique genes (not found in other databases) and carries functional labels (oncogene/tumour suppressor). Analysis of this dataset reveals that cancer lncRNAs are impacted by germline variants, somatic mutations and changes in expression consistent with inferred disease functions. Furthermore, we show how clinical/genomic features can be used to vet prospective gene sets from high-throughput sources. The combination of size and quality makes CLC2 a foundation for precision medicine, demonstrating cancer lncRNAs’ evolutionary and clinical significance.

A regulatory pathway that selectively up-regulates elongasome function in the absence of class A PBPs

Bacteria surround themselves with peptidoglycan, an adaptable enclosure that contributes to cell shape and stability. Peptidoglycan assembly relies on penicillin-binding proteins (PBPs) acting in concert with SEDS-family transglycosylases RodA and FtsW, which support cell elongation and division respectively. In Bacillus subtilis, cells lacking all four PBPs with transglycosylase activity (aPBPs) are viable. Here, we show that the alternative sigma factor σI is essential in the absence of aPBPs. Defects in aPBP-dependent wall synthesis are compensated by σI-dependent upregulation of an MreB homolog, MreBH, which localizes the LytE autolysin to the RodA-containing elongasome complex. Suppressor analysis reveals that cells unable to activate this σI stress response acquire gain-of-function mutations in the essential histidine kinase WalK, which also elevates expression of sigI, mreBH and lytE. These results reveal compensatory mechanisms that balance the directional peptidoglycan synthesis arising from the elongasome complex with the more diffusive action of aPBPs.

Cancer LncRNA Census 2 (CLC2): an enhanced resource reveals clinical features of cancer lncRNAs

Long noncoding RNAs play key roles in cancer and are at the vanguard of precision therapeutic development. These efforts depend on large and high-confidence collections of cancer lncRNAs. Here we present the Cancer LncRNA Census 2 (CLC2): at 492 cancer lncRNAs, it is 4-fold greater than its predecessor, without compromising on strict criteria of confident functional / genetic roles and inclusion in the GENCODE annotation scheme. This increase was enabled by leveraging high-throughput transposon insertional mutagenesis (TIM) screening data, yielding 95 novel cancer lncRNAs. CLC2 makes a valuable addition to existing collections: it is amongst the largest, holds the greatest number of unique genes, and carries functional labels (oncogene / tumour suppressor). Analysis of this dataset reveals that cancer lncRNAs are impacted by germline variants, somatic mutations, and changes in expression consistent with inferred disease functions. Furthermore, we show how clinical / genomic features can be used to vet prospective gene sets from high-throughput sources. The combination of size and quality makes CLC2 a foundation for precision medicine, demonstrating cancer lncRNAs’ evolutionary and clinical significance.

Phylogenetic debugging of a complete human biosynthetic pathway transplanted into yeast

Cross-species pathway transplantation enables insight into a biological process not possible through traditional approaches. We replaced the enzymes catalyzing the entire Saccharomyces cerevisiae adenine de novo biosynthesis pathway with the human pathway. While the ‘humanized’ yeast grew in the absence of adenine, it did so poorly. Dissection of the phenotype revealed that PPAT, the human ortholog of ADE4, showed only partial function whereas all other genes complemented fully. Suppressor analysis revealed other pathways that play a role in adenine de-novo pathway regulation. Phylogenetic analysis pointed to adaptations of enzyme regulation to endogenous metabolite level ‘setpoints’ in diverse organisms. Using DNA shuffling, we isolated specific amino acids combinations that stabilize the human protein in yeast. Thus, using adenine de novo biosynthesis as a proof of concept, we suggest that the engineering methods used in this study as well as the debugging strategies can be utilized to transplant metabolic pathway from any origin into yeast.

Coping with an Essential Poison: a Genetic Suppressor Analysis Corroborates a Key Function of c-di-AMP in Controlling Potassium Ion Homeostasis in Gram-Positive Bacteria

ABSTRACT Cyclic di-AMP (c-di-AMP) is an important second messenger in bacteria. In most Firmicutes , the molecule is required for growth in complex media but also toxic upon accumulation. In an article on their current study, Zarrella and coworkers present a suppressor analysis of a Streptococcus pneumoniae strain that is unable to degrade c-di-AMP (T. M. Zarrella, D. W. Metzger, and G. Bai, J Bacteriol 200:e00045-18, 2018, https://doi.org/10.1128/JB.00045-18 ). Their study identifies new links between c-di-AMP and potassium homeostasis and supports the hypothesis that c-di-AMP serves as a second messenger to report about the intracellular potassium concentrations.

Increased Activity of Cystathionine β-Lyase Suppresses 2-Aminoacrylate Stress inSalmonella enterica

ABSTRACTReactive enamine stress caused by intracellular 2-aminoacrylate accumulation leads to pleiotropic growth defects in a variety of organisms. Members of the well-conserved RidA/YER057c/UK114 protein family prevent enamine stress by enhancing the breakdown of 2-aminoacrylate to pyruvate. InSalmonella enterica, disruption of RidA allows 2-aminoacrylate to accumulate and to inactivate a variety of pyridoxal 5′-phosphate-dependent enzymes by generating covalent bonds with the enzyme and/or cofactor. This study was initiated to identify mechanisms that can overcome 2-aminoacrylate stress in the absence of RidA. Multicopy suppressor analysis revealed that overproduction of the methionine biosynthesis enzyme cystathionine β-lyase (MetC) (EC 4.4.1.8) alleviated the pleiotropic consequences of 2-aminoacrylate stress in aridAmutant strain. Degradation of cystathionine by MetC was not required for suppression ofridAphenotypes. The data support a model in which MetC acts on a noncystathionine substrate to generate a metabolite that reduces 2-aminoacrylate levels, representing a nonenzymatic mechanism of 2-aminoacrylate depletion.IMPORTANCERidA proteins are broadly conserved and have been demonstrated to deaminate 2-aminoacrylate and other enamines. 2-Aminoacrylate is generated as an obligatory intermediate in several pyridoxal 5′-phosphate-dependent reactions; if it accumulates, it damages cellular enzymes. This study identified a novel mechanism to eliminate 2-aminoacrylate stress that required the overproduction, but not the canonical activity, of cystathionine β-lyase. The data suggest that a metabolite-metabolite interaction is responsible for quenching 2-aminoacrylate, and they emphasize the need for emerging technologies to probe metabolismin vivo.